Proteins are the main building blocks and catalysts in life systems. Manipulation of genes through recombinant nucleic acid technologies, and expression of natural and engineered proteins, have provided many of the benefits associated with the genetic engineering revolution. Protein engineering, including incorporation of unnatural amino acids into peptides, can provide further practical benefits from the life sciences.
Translation of peptides encoded by nucleic acid sequences is accomplished in life systems through the complex interaction of many translation system constituent components, such as, e.g., ribosomes, mRNA, tRNAs, aminoacyl-tRNA synthetases, and amino acids. A strict set of rules and reliable reactions provide remarkably consistent translation of proteins by endogenous translation systems in living cells. A family of RNA polymerases first generates ribosomal RNA (rRNA), tRNAs, and mRNAs by transcription of DNA sequences. An endogenous family of aminoacyl-tRNA synthetases can each bind and link a specific amino acid (of the 20 natural amino acids) to a specific tRNA. Ribosomes, assembled from proteins and rRNA, align the unique anticodon of each tRNA with the complimentary codon presented in an mRNA chain to be translated. Finally, the ribosomes catalyze formation of a peptide bond between amino acids aligned together with their tRNAs along the mRNA chain. The ribosomes recognize a start codon (AUG-methionine) associated with a near by promoter sequence to determine a translation starting position and reading frame. Ribosomes generally respond to three mRNA termination codons (UAG, UGA, and UAA), not having associated tRNAs, as a signal to stop translation.
One way to provide proteins with unnatural side groups is to modify the protein after translation. Side groups of certain amino acids are chemically reactive and amenable to chemical modification. The sulfhydryl group of cysteine, hydroxyl group of tyrosine, and amino group of glutamine, e.g., can enter into reactions well known in the chemical arts, resulting in modifications or covalent bonding to side chains of amino acid residues. For example, lysine residue side chains, containing a epsilon-amino group, can be converted to acetyl-lysine by the enzymatic action of an acetyltransferase or by chemical reactions with, e.g., chemical acetylating agents, such as acetylacetate. However, post translational modifications are often non-specific and/or poorly directed.
Unnatural amino acids can also be incorporated into peptides by chemical synthesis. Automated chemical synthesis on a solid support matrix can provide a straightforward method to incorporate unnatural amino acids. However, routine solid-phase peptide synthesis is generally limited to small peptides or proteins with less than 100 residues. It is possible to make larger proteins with recently developed methods for enzymatic ligation or native chemical ligation of peptide fragments, but such methods are not easily scaled.
Unnatural amino acids can also be incorporated into proteins using mutant transcription system components. For example, orthogonal translation components can be added to native endogenous translation systems to translate peptides not normally provided by the endogenous translation system. In “An Engineered Escherichia coli Tyrosyl-tRNA Synthetase for Site-specific Incorporation of an Unnatural Amino Acid into Proteins in Eukaryotic Translation and Its Application in a Wheat Germ Cell-free System”, by A. K. Kowal, et al., Proc. Natl. Acad. Sci. USA 98, 2268-73 (2001), tyrosyl-tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-L-tyrosine rather than L-tyrosine for the site-specific incorporation of 3-iodo-L-tyrosine into proteins in eukaryotic in vitro translation systems. A similar translation system has been engineered to incorporate unnatural tyrosine analogs in a mammalian system. In “Site-specific Incorporation of an Unnatural Amino Acid into Proteins in Mammalian Cells”, by K. Sakamoto, N. A. Res., Vol. 30, No. 21 4692-4699, (2002), an E. Coli TyrRS construct was expressed along with an Bacillus stearothermophilus amber suppressor t-RNA in mammalian cells to provide a ras protein having an iodo-tyrosine residue encoded by a TAG codon. The system was specific to iodo-tyrosine incorporation, and failed to describe useful unique properties of the translated peptides.
In view of the above, a need exists for improved methods to specifically incorporate unnatural amino acid residues into peptides at desired positions using eukaryotic translation systems. It would be desirable to have a way to incorporate unnatural residues other than halogenated tyrosine residues to peptides. Benefits could also be realized through incorporation of unnatural amino acids that are detectable without tags. Methods for incorporation of unnatural amino acids having specifically reactive chemical linkage groups would be useful in the diagnostic, therapeutic and materials sciences. The present invention provides these and other features that will be apparent upon review of the following.